Connective Tissue Growth Factor Fragments and Methods and Uses Thereof

ABSTRACT

The present invention is directed to CTGF fragments comprising at least exon 2 or exon 3 of CTGF and having the ability to induce extracellular matrix synthesis, in particular, collagen synthesis and myofibroblast differentiation. The present invention is further directed to methods using said CTGF fragments to identify compositions which modulate the activity of said CTGF fragments and to the compositions so identified. The invention also relates to methods of treating CTGF-associated disorders and diseases associated with the overproduction of the extracellular matrix.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/315,568 filed Dec. 9, 2002, now pending; which is a divisionalapplication of U.S. application Ser. No. 09/461,688 filed Dec. 14, 1999,now issued as U.S. Pat. No. 6,492,129; which claims the benefit under 35USC§119(e) to U.S. Application Ser. No. 60/112,241 filed Dec. 14, 1998,now abandoned and to U.S. Application Ser. No. 60/112,240 filed Dec. 14,1998, now abandoned. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of growth factors andspecifically to fragments of Connective Tissue Growth Factor (CTGF) andmethods of use thereof.

2. Background Information

Growth Factors. Growth factors can be broadly defined asmultifunctional, locally acting intracellular signaling polypeptideswhich control both the ontogeny and maintenance of tissue form andfunction. The protein products of many proto-oncogenes have beenidentified as growth factors and growth factor receptors.

Growth factors generally stimulate target cells to proliferate,differentiate and organize in developing tissues. The action of growthfactors is dependent on their binding to specific receptors thatstimulate a signaling event within the cell. Examples of growth factorsinclude platelet derived growth factor (PDGF), insulin like growthfactor (IGF), transforming growth factor beta (TGF-β), transforminggrowth factor alpha (TGF-α), epidermal growth factor (EGF) andconnective tissue growth factor (CTGF). Each of these growth factors hasbeen reported to stimulate cells to proliferate.

Connective Tissue Growth Factor. CTGF is a cysteine rich monomericpeptide a molecular weight of about 38 kd. As previously reported, CTGFhas both mitogenic and chemotactic activities for connective tissuecells. CTGF is secreted by cells and is believed to be active uponinteraction with a specific cell receptor.

CTGF is a member of a family of growth regulators which include, forexample, mouse (fisp-12) and human CTGF, Cyr61 (mouse), Cef10 (chicken),and Nov (chicken). Based on sequence comparisons, is has been suggestedthat the members of this family have a modular structure consistingtypically of at least one of the following: (1) an insulin-like growthfactor domain responsible for binding; (2) a von Willebrand factordomain responsible for complex formation; (3) a thrombospondin type Irepeat, possibly responsible for binding matrix molecules; and (4) aC-terminal module found in matrix proteins, postulated to be responsiblefor receptor binding.

The sequence of the cDNA for human CGTF contains an open reading frameof 1047 nucleotides with an initiation site at about nucleotide 130 anda TGA termination site at about nucleotide 1177, and encodes a peptideof 349 amino acids. The cDNA sequence for human CTGF has been previouslydisclosed in U.S. Pat. No. 5,408,040.

The CTGF open reading frame encodes a polypeptide which contains 39cysteine residues, indicating a protein with multiple intramoleculardisulfide bonds. The amino terminus of the peptide contains ahydrophobic signal sequence indicative of a secreted protein and thereare two N-linked glycosylation sites at asparagine residues 28 and 225in the amino acid sequence.

The synthesis and secretion of CTGF are believed to be selectivelyinduced by TGF-β and BMP-2, as well as potentially by other members ofthe TGF-β superfamily of proteins. As reported in the art, althoughTGF-β can stimulate the growth of normal fibroblasts in soft agar, CTGFalone cannot induce this property in fibroblasts. However, it has beenshown that the synthesis and action of CTGF are essential for the TGF-βto stimulate anchorage independent fibroblast growth. See, e.g.,Kothapalli et al., 1997, Cell Growth & Differentation 8(1):61-68 andBoes et al., 1999, Endocrinology 140(4):1575-1580.

With respect to biological activity, CTGF has been reported to beprimarily mitogenic in nature (able to stimulate target cells toproliferate). CTGF has also been reported to have chemotactic activity.Pathologically, the full-length CTGF molecule has been reported to beinvolved in conditions where there is an overgrowth of connective tissuecells and overdeposition of the extracellular matrix. CTGF has also beendescribed in the art to be associated with conditions relating tovascular endothelial cell migration and proliferation, andneovascularization. The diseases and disorders relating to theseconditions, include, for example, fibrosis of the skin and major organs,cancer, and related diseases and disorders such as systemic sclerosis,angiogenesis, atherosclerosis, diabetic nephropathy, and renalhypertension. (See, e.g., Toshifumi et al., 1999, Journal of CellularPhysiology 18191):153-159; Shimo et al., 1999, Journal of Biochemistry126(1):137-145; Murphy et al., 1999, Journal of Biological Chemistry274(9):5830-5834; Wenger et al., 1999, Oncogene 18(4):1073-1080; Frazieret al., 1997, International Journal of Biochemistry & Cell Biology29(1); 153-161; Oemar et al., 1997, Circulation 95(4); 831-839.)

CTGF has also been reported to be useful in wound healing and repair ofconnective tissue, bone and cartilage. In this aspect, CTGF has beendescribed as an inducer of bone, tissue, or cartilage formation indisorders such as osteoporosis, osteoarthritis or osteochondrytis,arthritis, skeletal disorders, hypertrophic scars, burns, vascularhypertrophy or wound healing. (See, e.g., U.S. Pat. No. 5,837,258;Ohnishi et al., 1998, Journal of Molecular and Cellular Cardiology30(10:2411-2422; Nakanishi et al., 1997, Biochemical and BiophysicalResearch Communications 234(1):206-210; Pawar et al., 1995, Journal ofCellular Physiology 165(3):556-565.)

In summary, CTGF has been implicated in numerous fibrotic and cancerousconditions, and has been described to contribute to wound healing. As aresult, there is a need in the art to identify useful methods ofmodulating the activity of CTGF to treat these various diseases anddisorders. Prior to the present invention, there has been no report thatregions or domains of CTGF are responsible for signaling differentbiological activities. Moreover, prior to the instant invention, therehas been no disclosure of treating diseases and disorders associatedwith cell proliferation and/or the overproduction of the extracellularmatrix by inhibiting the biological activity of a specific region ordomain of CTGF.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods for thetreatment of CTGF-associated diseases, disorders or ailments wherein thedeposition of the extracellular matrix is implicated, including, forexample, the induction of collagen synthesis and myofibroblastdifferentiation. More specifically, the compositions of the presentinvention comprise CTGF fragments comprising the N-terminal region ofCTGF. In one aspect, the fragment of the present invention comprises atleast a part of exons 2 or 3, or the polypeptide encoded thereby, is notthe CTGF fragment disclosed in Brigstock et al., 1997, J. Biol. Chem.272(32):20275-82, and further possesses the ability to induce synthesisof the extracellular matrix, including, but not limited to, the abilityto induce collagen, and myfibroblast differentiation. In a furtheraspect, the fragment of the present invention comprises between aboutone-quarter and one-half the length of the full-length CTGF protein.

In one aspect, a fragment of connective tissue growth factor (CTGF)polypeptide having the activities as described above is provided. Afragment of the invention includes CTGF having an amino acid sequenceencoded by at least exon 2 as set forth in FIG. 3. A fragment may alsoinclude an amino acid sequence encoded by at least exon 3 as set forthin FIG. 3. Further, a CTGF fragment of the invention may include anamino acid sequence encoded by at least exons 2 and 3 as set forth inFIG. 3. The invention also provides polynucleotide sequences encodingsuch fragments.

The present invention further comprises methods of using the CTGFfragments of the disclosed invention to identify compositions which canmodulate the activity of said CTGF fragments, wherein such compositionsmay be used to control normal deposition of the extracellular matrix,such as collagen deposition, as desired. More specifically, the CTGFfragments may be used to identify compositions that may control normaldeposition of collagen deposition and myfibroblast differentiation,wherein such composition may be used to inhibit, suppress, or increasethe activity of the CTGF fragments of the present invention.

The compositions of the claimed invention further comprise CTGFmodulators, for example, antibodies, antisense molecules, smallmolecules, and other compounds identified by the above methods, whichcan modulate the activity of the CTGF fragments of the presentinvention. In one aspect, the present invention provides CTGF modulatorsthat inhibit or suppress the activity of CTGF or the CTGF fragments. Inanother aspect of the present invention, the CTGF modulators increasethe activity of CTGF or the CTGF fragments, for example, in indicationswherein the induction of CTGF activity is desirable, for example, inwound healing, tissue repair, and bone repair.

In another aspect of the invention, the methods of the present inventioncomprise the administration of an effective amount of the CTGF fragmentmodulators, alone or in combination with one or more compounds, to apatient in need to treat diseases, disorders or ailments wherein themanipulation of collagen synthesis is desired. More particularly, themethods of the instant invention are directed to utilizing the compoundscapable of modulating the activity of the CTGF fragments of thisinvention to modulate collagen synthesis, and consequently, treatdisorders related to the overabundance of collagen synthesis, includingfibrotic disorders. Preferably, the disorders are of the dermis, themajor organ and disorders related to the overproduction of scar tissue.

The present invention also provides pharmaceutical compositionscomprising the CTGF fragments of the present invention. Suchcompositions may be useful in wound healing, bone and tissue repair,wherein the increased activity of CTGF is desirable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cells in a myoblast induction assay.

FIG. 2 shows a graph indicating that CTGF fragments of the presentinvention induce collagen synthesis in NRK cells.

FIG. 3 sets forth the nucleic acid sequence (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of the full length CTGF molecule, whereinthe location of each exon of the CTGF molecule is identified.

FIG. 4 sets forth the nucleic acid sequence (SEQ ID NO:3) and the aminoacid sequence (SEQ ID NO:4) of the N-terminal domain of CTGF comprisingexon 2 and exon 3 of the CTGF molecule.

FIG. 5 sets forth data relating to the inhibition of collagen synthesiswith anti-CTGF antibodies.

FIG. 6 sets forth data relating to the inhibition of collagen synthesisby antibodies directed to the N-terminal domain of CTGF.

FIG. 7 sets forth data relating to the stimulation of collagen synthesisby CTGF and the N-terminal domain of CTGF.

FIG. 8 sets forth data relating to the N-terminal domain of CTGF as anactive inducer of collagen synthesis and myofibroblast induction.

FIG. 9 sets forth data relating to the effects of IGF-2 on CTGF-inducedcollagen synthesis.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagents,etc., described herein, as these may vary. It is also to be understoodthat the terminology used herein is used for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention. It must be noted that as used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “an antibody” is a reference to one or moreantibodies and equivalents thereof known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Preferred methods, devices,and materials are described, although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention. All references cited herein areincorporated by reference herein in their entirety.

DEFINITIONS

As used herein, the term “CTGF fragment” refers to a fragment comprisingat least a part of the N-terminal region of CTGF. In one embodiment, thefragment comprises at least a part of exons 2 or 3 of the full lengthCTGF protein, and further possesses the ability to induce synthesis ofthe extracellular matrix. In another embodiment, the fragment is betweenabout one-quarter and one-half the length of the full-length CTGFprotein. “The ability to induce collagen synthesis” shall mean theability to induce the formation of the extracellular matrix via collagensynthesis and myofibroblast differentiation. The CTGF fragments may beeither obtained by isolation from natural sources, synthetic manufactureproduction, recombinant genetic engineering techniques, or othertechniques available in the art.

As used herein, the term “N-terminal” refers to the nucleic acidsequence comprising at least part of the exon 2 and exon 3 domains,beginning at about of the full length CTGF molecule, and to the encodedamino acid sequence, as identified in FIGS. 3A and 3B. The term “exon 2”refers to the nucleic acid sequence and corresponding amino acidsequence of the N-terminal domain, beginning at about of the full lengthCTGF molecule, and the corresponding amino acid sequence. The term “exon3”, refers to the nucleic acid sequence of the N-terminal domain of thefull length CTGF molecule and to the encoded polypeptide.

As used herein, the term “C-terminal” refers to the nucleic acidsequence comprising at least a part of the exon 4 and exon 5 domains ofthe full length CTGF molecule, and to the polypeptide encoded thereby,as identified in FIGS. 3A and 3B.

The terms “disorders” and “diseases” as used herein, refers toconditions associated with the expression or activity of CTGF. Diseases,disorders, and conditions associated with CTGF include, but are notlimited to, excessive scarring resulting from acute or repetitivetraumas, including surgery or radiation therapy, fibrosis of organs suchas the kidney, lungs, liver, eyes, heart, and skin, includingscleroderma, keloids, and hypertrophic scarring. Abnormal expression ofCTGF has been associated with general tissue scarring, tumor-likegrowths in the skin, and sustained scarring of blood vessels, leading toimpaired blood-carrying ability, hypertension, hypertrophy, etc. Alsoassociated with CTGF are various diseases caused by vascular endothelialcell proliferation or migration, such as cancer, includingdermatofibromas, conditions related to abnormal endothelial cellexpression, breast carcinoma desmosplasis, angiolipoma, andangioleiomyoma. Other related conditions include atherosclerosis andsystemic sclerosis, including atherosclerotic plaques, inflammatorybowel disease, Crohn's disease, angiogenesis, and other proliferativeprocesses which play central roles in atherosclerosis, arthritis,cancer, and other disease states, neovascularization involved inglaucoma, inflammation due to disease or injury, including jointinflammation, tumor growth metastasis, interstitial disease,dermatological diseases, arthritis, including chronic rheumatoidarthritis, arteriosclerosis, diabetes, including diabetic nephropathy,hypertension, and other kidney disorders, and fibrosis resulting fromchemotherapy, radiation treatment, dialysis, and allograft andtransplant rejection.

“Fibroproliferative” disorders as referred to herein include but are notlimited to any of the diseases or disorders listed above, for example,kidney fibrosis, scleroderma, pulmonary fibrosis, arthritis, hypertropicscarring, and atherosclerosis. CTGF-associated fibroproliferativedisorders also include diabetic nephropathy and retinopathy,hypertension, and other kidney disorders, angiogenesis-relateddisorders, including but not limited to blood vessels associated withtumor formation, and other proliferative processes which play centralroles in atherosclerosis, arthritis, and other disease states, and, forexample, in skin, cardiac, and pulmonary and renal fibrosis. In general,severe fibrosis involving kidney, liver, lung, and the cardiovascularsystem are included herein. There are numerous examples of fibrosis,including the formation of scar tissue following a heart attack, whichimpairs the ability of the heart to pump. Diabetes frequently causesdamage/scarring in the kidneys which leads to a progressive loss ofkidney function. Even after surgery, scar tissue can form betweeninternal organs causing contracture, pain, and, in some cases,infertility. Major organs such as the heart, kidney, liver, lung, eye,and skin are prone to chronic scarring, commonly associated with otherdiseases. Hypertrophic scars (non-malignant tissue bulk) are a commonform of fibrosis caused by burns and other trauma. In addition, thereare a number of other fibroproliferative disorders such as scleroderma,keloids, and atherosclerosis which are associated respectively withgeneral tissue scarring, tumor-like growths in the skin, or a sustainedscarring of blood vessels which impairs blood carrying ability. As CTGFis overexpressed in fibrotic disorders, it represents a very specifictarget for the development of anti-fibrotic therapeutics. CTGF can beinhibited through the use of small molecules and neutralizingantibodies, for example, in the treatment of fibroproliferativedisorders. It is understood that “fibroproliferative” refers to any ofthe above pathological instances and should not be limited to cellularproliferation.

“Extracellular matrices (ECM)” are multi-component structuressynthesized by and surrounding various cell types including, forexample, endothelial, epithelial, epidermal, and muscle cells. The ECMis formed largely of collagen and heparin sulfate proteoglycans. The ECMalso contains hlfibronectin, vitronectin, chondroitin sulfateproteoglycans, and smaller proteins. Growth factors are sequestered inthese matrices by association with the glycosaminoglycan portion of theheparin sulfate proteoglycans. “Heparin like” regions of high iduronicacid and high sulfation have been associated with the bFGF bindingregion of heparin sulfate from human fibroblasts (Turnbull, et al., J.Biol. Chem. 267(15) 10337-10341, 1992). However, the composition ofheparin sulfate in the extracellular matrix has not been fullycharacterized.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature, andare well known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “substantial amino acid homology” refers to molecules having asequence similarity of approximately 75% or more, preferably 85% or moreand more preferably 90-95% to a specific sequence. The phrases “%similarity” or “% identity” refer to the percentage of sequencesimilarity or identity found in a comparison of two or more amino acidor nucleic acid sequences. Percent similarity can be determined bymethods well-known in the art.

Percent similarity between amino acid sequences can be calculated, forexample, using the clustal method. (See, e.g., Higgins, D. G. and P. M.Sharp, 1988, Gene 73:237-244.) The clustal algorithm groups sequencesinto clusters by examining the distances between all pairs. The clustersare aligned pairwise and then in groups. The percentage similaritybetween two amino acid sequences, e.g., sequence A and sequence B, iscalculated by dividing the length of sequence A, minus the number of gapresidues in sequence A, minus the number of gap residues in sequence B,into the sum of the residue matches between sequence A and sequence B,times one hundred. Gaps of low or of ho homology between the two aminoacid sequences are not included in determining percentage similarity.Percent similarity can be calculated by other methods known in the art,for example, by varying hybridization conditions, and can be calculatedelectronically using programs such as the MegAlign program. (DNASTARInc., Madison, Wis.)

The term “collagen subunit” refers to the amino acid sequence of onepolypeptide chain of a collagen protein encoded by a single gene, aswell as any derivatives that sequence, including deletion derivatives,conservative substitutions, etc.

A “fusion protein” is a protein in which peptide sequences fromdifferent proteins are covalently linked together.

An “antisense sequence” is any sequence capable of specificallyhybridizing to a target sequence. The antisense sequence can be DNA,RNA, or any nucleic acid mimic or analog. The term “antisensetechnology” refers to any technology which relies on the specifichybridization of an antisense sequence to a target sequence.

The term “functional equivalent”, as used herein refers to a protein ornucleic acid molecule that possesses functional or structuralcharacteristics to CTGF fragment. A functional equivalent of a CTGFfragment may contain modifications depending on the necessity of suchmodifications for the performance of a specific function. The term“functional equivalent” is intended to include fragments, mutants,hybrids, variants, analogs, or chemical derivatives of a molecule.

A molecule is said to be a “chemical derivative” of another moleculewhen it contains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, and the like. The moieties canalternatively decrease the toxicity of the molecule, eliminate orattenuate any undesirable side effect of the molecule, and the like.Moieties capable of mediating such effects are disclosed, for example,in Gennaro, A. R., ed, 1990, Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton Pa. Procedures for coupling suchmoieties to a molecule are well known in the art.

A “variant,” as used herein, refers to an amino acid sequence that isaltered by one or more amino acids. The variant may have conservativechanges, wherein a substituted amino acid has similar structural orchemical properties, e.g., replacement of leucine with isoleucine. Morerarely, a variant may have nonconservative changes, e.g., replacement ofa glycine with a tryptophan. Analogous minor variations may also includeamino acid deletions or insertions, or both. Guidance in determiningwhich amino acid residues may be substituted, inserted, or deleted maybe found using computer programs well known in the art, for example,DNASTAR software (DNASTAR Inc., Madison, Wis.).

Methods for Making CTGF Fragments

Nucleic Acid Sequences Encoding CTGF. In accordance with the invention,nucleotide sequences encoding CTGF or functional equivalents thereof, asdescribed in U.S. Pat. No. 5,408,040, may be used to generaterecombinant DNA molecules that direct the expression of the full lengthprotein or a functional equivalent thereof, or alternatively, nucleotidesequences encoding the desired CTGF fragment, for example, inappropriate host cells, a fragment comprising at least a part of exons 2or 3 of CTGF.

Alternatively, nucleotide sequences which hybridize, under stringentposition, to portions of the CTGF sequence may also be used in nucleicacid hybridization assays, Southern and Northern blot analyses, etc. Inyet another method, DNA molecules encoding CTGF may be isolated byhybridization procedures comprising antibody screening of expressionlibraries to detect shared structural features.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode proteins of substantial amino acid homology or afunctionally equivalent amino acid sequence, may be isolated and used inthe practice of the invention for the cloning and expression of CTGF orthe CTGF fragment. Such DNA sequences include those which are capable ofhybridizing to the human CTGF sequence under stringent conditions.

Altered DNA sequences which may be used in accordance with the inventioninclude deletions, additions or substitutions of different nucleotideresidues resulting in a sequence that encodes the same or a functionallyequivalent gene product. The gene product itself may contain deletions,additions or substitutions of amino acid residues within the CTGFsequence, which result in a silent change thus producing a functionallyequivalent protein. Such amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipatic nature of the residues involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine; glycine, analine; asparagine, glutamine; serine, threonine;phenylalanine, tyrosine.

The DNA sequences of the invention may be engineered in order to alterthe protein's sequence for a variety of ends including but not limitedto alterations which modify processing and expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, e.g., site-directed mutagenesis to, forexample, insert new restriction sites. For example, in certainexpression systems such as yeast, host cells may over-glycosylate thegene product. When using such expression systems it may be preferable toalter CTGF or CTGF fragment coding sequence to eliminate any N-linkedglycosylation site.

The CTGF or CTGF fragment sequence may be ligated to a heterologoussequence to encode a fusion protein. For example, for screening ofpeptide libraries it may be useful to encode a chimeric CTGF proteinexpressing a heterologous epitope that is recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the CTGF or CTGF fragment sequence and theheterologous protein sequence (e.g. a sequence encoding a growth factorrelated to PDGF), so that CTGF or a CTGF fragment can be cleaved awayfrom the heterologous moiety. Such methods are known in the art.

The coding sequence of CTGF or a CTGF fragment may also be synthesizedin whole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, et al., 1980, Nucleic Acids Res. Symp. Ser. 7:215-233;Crea and Horn, 1980, Nucleic Acids Res. 9(10):2331; Matteucci andCaruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981,Nucleic Acids Res. 9(12):2807-2817.) Alternatively, the protein itselfcould be produced using chemical methods to synthesize the CTGF aminoacid sequence in whole or in part. For example, peptides can besynthesized by solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography. Seee.g., Creighton, 1983, Proteins Structures And Molecular Principles,W.H. Freeman and Co., N.Y. pp. 50-60. The composition of the syntheticpeptides may be confirmed by amino acid analysis or sequencing. Forexample, for the Edman degradation procedure, see, Creighton, 1983,Proteins, Structures and Molecular Principles, W.H. Freeman and Co.,N.Y., pp. 34-49.

Expression Of CTGF Or A CTGF Fragment. In order to express abiologically active CTGF fragment, the nucleotide sequence coding forthe full length protein, or a functional equivalent as described above,the CTGF fragment is inserted into an appropriate expression vector,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence.

More specifically, methods which are well known to those skilled in theart can be used to construct expression vectors containing the CTGF orCTGF fragment sequence and appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See e.g., the techniques described in Maniatis et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. and Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y.

A variety of host-expression vector systems may be utilized to expressthe CTGF or CTGF fragment coding sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the CTGF or CTGF fragment coding sequence; yeast, includingPichia pastoris and Hansenula polymorphs, transformed with recombinantyeast expression vectors containing the CTGF or CTGF fragment codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., bacculovirus) containing the CTGF or CTGF fragment codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing the CTGF and CTGF fragment coding sequence; oranimal cell systems infected with recombinant virus expression vectors(e.g., adenovirus, vaccinia virus, human tumor cells (includingHT-1080)) including cell lines engineered to contain multiple copies ofthe CTGF DNA either stably amplified (CHO/dhfr) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines). As used herein, itis understood that the term “host-expression vector systems” and moregenerally, the term “host cells” includes any progeny of the host cellor host-expression vector system. It is further understood that althoughall progeny may not be identical to the parental cell, as mutations mayoccur during replication, such progeny are included in the scope of theinvention.

The expression elements of these systems vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the bacculovirus polyhedrin promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used;when generating cell lines that contain multiple copies of the CTGF orCTGF fragment DNA SV40-, BPV- and EBV-based vectors may be used with anappropriate selectable marker.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for theexpressed CTGF or CTGF fragment. For example, a suitable vector forexpression in bacteria includes the T7-based vector as described inRosenberg, et al., 1987, Gene 56:125. As further example, when largequantities of CTGF or CTGF fragment are to be produced to screen peptidelibraries, vectors which direct the expression of high levels of proteinproducts that are readily purified may be desirable. Such vectorsinclude but are not limited to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which the CTGF or CTGFfragment coding sequence may be ligated into the vector in frame withthe lac Z coding region so that a hybrid AS-lac Z protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides such asCTGF or a CTGF fragment with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety.

More generally, where the host is a prokaryote, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth and subsequently treated by the CaCl₂, oralternatively MgCl₂ or RbC1, method using procedures well known in theart.

Where the host cell is a eukaryote, various methods of DNA transfer canbe used. These include transfection of DNA by calciumphosphate-precipitates, conventional mechanical procedures, includingmicroinjection, insertion of a plasmid encased in liposomes, or use ofvirus vectors. Eukaryotic cells may also be cotransformed with DNAsequences encoding the polypeptide of the invention, and a secondforeign DNA molecule encoding a selectable phenotype, such as herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eucaryotic cells and express protein.See, Eukaryotic Viral Vectors, 1992, Cold Spring Harbor Laboratory,Gluzman, Ed.). Eucaryotic host cells include yeast, mammalian cells,insect cells and plant cells.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review, see, e.g., Current Protocols inMolecular Biology, Vol. 2, 1988, Ausubel et al., Ed., Greene Publish.Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Methods inEnzymology, Wu & Grossman, Eds., Acad. Press, N.Y., 153:516-544; Glover,1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; Bitter, 1987,Heterologous Gene Expression in Yeast, Methods in Enzymology, Berger &Kimmel, Eds., Acad. Press, N.Y., 152:673-684; and The Molecular Biologyof the Yeast Saccharomyces, 1982, Strathern et al., Eds., Cold SpringHarbor Press, Vols. I and II. For example, various shuttle vectors forthe expression of foreign genes in yeast have been reported. Heinemann,et al., 1989, Nature 340:205; Rose, et al., 1987, Gene 60:237.

In cases where plant expression vectors are used, the expression of theCTGF or CTGF fragment coding sequence may be driven by any of a numberof promoters. For example, viral promoters such as the 35S RNA and 19SRNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or thecoat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311)may be used; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al.,1984, Science 224:838-843); or heat shock promoters, e.g., soybeanhsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565)may be used. These constructs can be introduced into plant cells usingTi plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. For reviews ofsuch techniques, see, e.g., Weissbach & Weissbach, 1988, Methods forPlant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463;Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9.

In an insect system, an alternative expression system could be used toexpress CTGF or a CTGF fragment. In one such system, Bacculovirus isused as a vector to express foreign genes. The virus then grows in theinsect cells. The CTGF or CTGF fragment coding sequence may be clonedinto non-essential regions (for example the polyhedrin gene) of thevirus and placed under control of a Bacculovirus promoter. Theserecombinant viruses are then used to infect insect cells in which theinserted gene is expressed. See, e.g., Smith et al., 1983, J. Virol.46:584; Smith, U.S. Pat. No. 4,215,051.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the CTGF or CTGF fragment coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing CTGF or a CTGF fragment in infected hosts. See e.g., Logan &Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659. Alternatively,the vaccinia 7.5K promoter may be used. See, e.g., Mackett et al., 1982,Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J.Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci.79:4927-4931.

In another embodiment, the CTGF or CTGF fragment sequence is expressedin human tumor cells, such as HT-1080, which have been stablytransfected with calcium phosphate precipitation and a neomycinresistance gene. In yet another embodiment, the pMSXND expression vectoror the like is used for expression in a variety of mammalian cells,including COS, BHK 293 and CHO cells. Lee and Nathans, 1988, J. Biol.Chem. 263:3521.

Specific initiation signals may also be required for efficienttranslation of inserted CTGF or CTGF fragment coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where the entire CTGF gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. However, incases where only a portion of the CTGF coding sequence is inserted,exogenous translational control signals, including the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the CTGF or CTGF fragment codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. See e.g., Bitter et al., 1987,Methods in Enzymol. 153:516-544. Additional sequences, i.e., leadersequences, etc., may be added to direct the secretion of CTGF or CTGFfragments along various secretory pathways. This can be accomplished ina number of expression systems using various methods known in the art.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, HT-1080, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressCTGF or CTGF fragment may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with CTGF or CTGF fragment DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.) and a selectablemarker. Following the introduction of foreign DNA, engineered cells maybe allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. (USA) 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk, hgprt or aprt cells, respectively.

Also, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler, et al.,1980, Proc. Natl. Acad. Sci. (USA) 77:3567; O′Hare, et al., 1981, Proc.Natl. Acad. Sci. (USA) 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. (USA)78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147)genes. Recently, additional selectable genes have been described, namelytrpB, which allows cells to utilize indole in place of tryptophan; hisD,which allows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. (USA) 85:8047); and ODC(ornithine decarboxylase) which confers resistance to the ornithinedecarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue, 1987, In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory).

The isolation and purification of host cell-expressed polypeptides ofthe invention may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibody.

Identification Of Transfectants Or Transformants That Express CTGF Or ACTGF Fragment. The host cells which contain the coding sequence andwhich express the biologically active gene product may be identified byat least four general approaches: (a) DNA-DNA or DNA-RNA hybridization;(b) the presence or absence of “marker” gene functions; (c) assessingthe level of transcription as measured by the expression of CTGF or CTGFfragment mRNA transcripts in the host cell; and (d) detection of thegene product as measured by an assay or by its biological activity.

In the first approach, the presence of the CTGF or CTGF fragment codingsequence inserted in the expression vector can be detected by DNA-DNA orDNA-RNA hybridization using probes comprising nucleotide sequences thatare homologous to the CTGF or CTGF fragment coding sequence,respectively, or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., resistance to antibiotics,resistance to methotrexate, transformation phenotype, occlusion bodyformation in bacculovirus, etc.). For example, in a preferredembodiment, the CTGF coding sequence is inserted within aneomycin-resistance marker gene sequence of the vector, and recombinantscontaining the CTGF coding sequence can be identified by the absence ofthe marker gene function. Alternatively, a marker gene can be placed intandem with the CTGF sequence under the control of the same or differentpromoter used to control the expression of the CTGF coding sequence.Expression of the marker in response to induction or selection indicatesexpression of the CTGF coding sequence.

In the third approach, transcriptional activity for the CTGF or CTGFfragment coding region can be assessed by hybridization assays. Forexample, RNA can be isolated and analyzed by northern blot using a probehomologous to the CTGF or CTGF fragment coding sequence or particularportions thereof. Alternatively, total nucleic acids of the host cellmay be extracted and assayed for hybridization to such probes.

The fourth approach involves the detection of the biologically active orimmunologically reactive CTGF or CTGF fragment gene product. A number ofassays can be used to detect CTGF activity including, but not limitedto, those assays described in U.S. Pat. No. 5,408,040.

Cleavage of Full-Length CTGF Protein To Produce CTGF Fragment. Followingexpression of the full length CTGF protein, the protein may be cleavedby any number of proteolytic enzymes known to one of ordinary skill inthe art to result in the CTGF fragments of the present invention. Forexample, the cysteine free bridge between the N-terminal and C-terminalhalves of CTGF are susceptible to chymotrypsin using methods availablein the art.

Methods for Modulating and Inhibiting Activity of CTGF Fragments

As described above, the CTGF fragments described in this invention are acritical determinant of extracellular matrix deposition in fibroticconditions. The present invention provides for methods for theprevention and treatment of complications associated with the activityof said fragments by regulating, modulating, and/or inhibiting theactivity and or expression of such fragments, or if desirable increasingthe activity of such fragments. More specifically, methods of thepresent invention provide for the administration of a therapeuticallyeffective amount of an agent that regulates, modulates, and/or inhibitsthe extracellular matrix producing activity of the N-terminal fragmentsof CTGF, as desired, to treat, prevent or ameliorate diseases, ordisorders associated with the expression and activity of CTGF.

Antibodies. In one embodiment of the present invention, a methodinvolves the administration of a therapeutically effective amount of anantibody which specifically reacts with the CTGF fragments of thepresent invention. Antibodies specifically reactive with CTGF aredescribed in U.S. Pat. No. 5,783,187 and PCT publication, WO 9638172.CTGF antibodies may be generated using methods well known in the art.Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain antibodies, as well as Fab fragments,including F(ab′)₂ and F_(v) fragments. Fragments can be produced, forexample, by a Fab expression library. Neutralizing antibodies, i.e.,those which inhibit dimer formation, are especially preferred fortherapeutic use.

A target polypeptide, such as CTGF or an agent that modulates theactivity and or expression of CTGF, can be evaluated to determineregions of high immunogenicity. Methods of analysis and epitopeselection are well-known in the art. See, e.g., Ausubel, et al., eds.,1988, Current Protocols in Molecular Biology. Analysis and selection canalso be accomplished, for example, by various software packages, such asLASERGENE NAVIGATOR software. (DNASTAR; Madison Wis.) The peptides orfragments used to induce antibodies should be antigenic, but are notnecessarily biologically active. Preferably, an antigenic fragment orpeptide is at least 5 amino acids in length, more preferably, at least10 amino acids in length, and most preferably, at least 15 amino acidsin length. It is preferable that the antibody-inducing fragment orpeptide is identical to at least a portion of the amino acid sequence ofthe target polypeptide, e.g., CTGF. A peptide or fragment that mimics atleast a portion of the sequence of the naturally occurring targetpolypeptide can also be fused with another protein, e.g., keyhole limpethemocyanin (KLH), and antibodies can be produced against the chimericmolecule.

Methods for the production of antibodies are well-known in the art. Forexample, various hosts, including goats, rabbits, rats, mice, humans,and others, may be immunized by injection with the target polypeptide orany immunogenic fragment or peptide thereof. Depending on the hostspecies, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund'sadjuvant, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable.

Monoclonal and polycolonal antibodies may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Techniques for in vivo and in vitroproduction are well-known in the art. See, e.g., Pound, J. D., 1998,Immunochemical Protocols, Humana Press, Totowa N.J.; Harlow, E. and D.Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, New York. The production of chimeric antibodies is alsowell-known, as is the production of single-chain antibodies. See, e.g.,Morrison, S. L. et al., 1984, Proc. Natl. Acad. Sci. 81:6851-6855;Neuberger, M. S. et al., 1984, Nature 312:604-608; Takeda, S. et al.,1985 Nature 314:452-454. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated, for example, by chainshuffling from random combinatorial immunoglobin libraries. See, e.g.,Burton D. R., 1991, Proc. Natl. Acad. Sci. 88:11120-11123.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents. See, e.g., Orlandi, R. et al.,1989, Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. and C. Milstein,1991, Nature 349:293-299). Antibody fragments which contain specificbinding sites for the target polypeptide may also be generated. Suchantibody fragments include, but are not limited to, F(ab′)₂ fragments,which can be produced by pepsin digestion of the antibody molecule, andFab fragments, which can be generated by reducing the disulfide bridgesof the F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. See, e.g., Huse, W. D., et al.,1989 Science 254:1275-1281.

Antibodies can be tested for anti-target polypeptide activity using avariety of methods well-known in the art. Various techniques may be usedfor screening to identify antibodies having the desired specificity,including various immunoassays, such as enzyme-linked immunosorbentassays (ELISAs), including direct and ligand-capture ELISAs,radioimmunoassays (RIAs), immunoblotting, and fluorescent activated cellsorting (FACS). Numerous protocols for competitive binding orimmunoradiometric assays, using either polyclonal or monoclonalantibodies with established specificities, are well known in the artSee, e.g., Harlow and Lane. Such immunoassays typically involve themeasurement of complex formation between the target polypeptide and aspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on thetarget polypeptide is preferred, but other assays, such as a competitivebinding assay, may also be employed. See, e.g., Maddox, D. E., et al.,1983, J Exp Med 158:1211.

The present invention contemplates the use of antibodies specificallyreactive with a CTGF polypeptide or fragments thereof which neutralizethe biological activity of the CTGF fragments of the present invention.The antibody administered in the method can be the intact antibody orantigen binding fragments thereof, such as Fab, F(ab′)₂ and F_(v)fragments, which are capable of binding the epitopic determinant. Theantibodies used in the method can be polyclonal or, more preferably,monoclonal antibodies. Monoclonal antibodies with different epitopicspecificities are made from antigen containing fragments of the proteinby methods well known in the art. See, e.g., Kohler et al., Nature256:494; Ausubel, et al., supra.

In the present invention, therapeutic applications include those using“human” or “humanized” antibodies directed to CTGF or fragments thereof.Humanized antibodies are antibodies, or antibody fragments, that havethe same binding specificity as a parent antibody, (i.e., typically ofmouse origin) and increased human characteristics. Humanized antibodiesmay be obtained, for example, by chain shuffling or by using phagedisplay technology. For example, a polypeptide comprising a heavy orlight chain variable domain of a non-human antibody specific for a CTGFis combined with a repertoire of human complementary (light or heavy)chain variable domains. Hybrid pairings specific for the antigen ofinterest are selected. Human chains from the selected pairings may thenbe combined with a repertoire of human complementary variable domains(heavy or light) and humanized antibody polypeptide dimers can beselected for binding specificity for an antigen. Techniques describedfor generation of humanized antibodies that can be used in the method ofthe present invention are disclosed in, for example, U.S. Pat. Nos.5,565,332; 5,585,089; 5,694,761; and 5,693,762. Furthermore, techniquesdescribed for the production of human antibodies in transgenic mice aredescribed in, for example, U.S. Pat. Nos. 5,545,806 and 5,569,825.

Antisense Oligonucleotides. The present invention provides for atherapeutic approach which directly interferes with the translation ofCTGF messages, and specifically the messages of the full length CTGF(wherein the full length protein is then cleaved to form a CTGF fragmentof the present invention) or the fragment of CTGF (collectively “CTGFmRNA”), into protein. More specifically, the present invention providesa method wherein antisense nucleic acid or ribozymes are used to bind toor cleave CTGF mRNA. Antisense RNA or DNA molecules bind specificallywith a targeted gene's RNA message, interrupting the expression of thatgene's protein product. The antisense binds to the messenger RNA forminga double stranded molecule which cannot be translated by the cell. Inaddition, chemically reactive groups, such as iron-linkedethylenediaminetetraacetic acid (EDTA-Fe) can be attached to anantisense oligonucleotide, causing cleavage of the RNA at the site ofhybridization. These and other uses of antisense methods to inhibit thetranslation of genes are well known in the art. See, for example,Marcu-Sakura, 1988, Anal. Biochem 177:278.

More specifically, in one embodiment of the invention, the sequence ofan antisense polynucleotide useful for inhibiting expression of the CTGFmRNA can be obtained by comparing the sequences of orthologous genes(sequences that are conserved between species), or the transcripts oforthologous genes, and identifying highly conserved regions within suchsequences. Similarity in nucleic acid sequences may be determined byprocedures and algorithms which are well-known in the art. Suchprocedures and algorithms include, for example, a BLAST program (BasicLocal Alignment Search Tool at the National Center for BiologicalInformation), ALIGN, AMAS (Analysis of Multiple Aligned Sequences), andAMPS (Protein Multiple Sequence Alignment).

In selecting the preferred length for a given polynucleotide, variousfactors should be considered to achieve the most favorablecharacteristics. In one aspect, polynucleotides of the present inventionare at least 15 base pairs (bp) in length and preferably about 15 toabout 100 by in length. More preferably, the polynucleotides are about15 by to about 80 by in length and even more preferably, thepolynucleotides of the present invention are about 15 to about 60 by inlength. Shorter polynucleotides, such as 10 to under 15-mers, whileoffering higher cell penetration, have lower gene specificity. Incontrast, longer polynucleotides of 20 to about 30 by offer betterspecificity, and show decreased uptake kinetics into cells. See, Steinet al., “Oligodeoxynucleotides: Antisense Inhibitors of GeneExpression,” Cohen, ed., McMillan Press, London (1988). Accessibility totranscript RNA target sequences also is of importance loop-formingregions and orthologous sequences in targeted RNAs thus offer promisingtargets. In this disclosure, the term “polynucleotide” encompasses botholigomeric nucleic acid moieties of the type found in nature, such asdeoxyribonucleotide and ribonucleotide structures of DNA and RNA, andman-made analogues which are capable of binding to nucleic acid found innature. The polynucleotides of the present invention can be based uponribonucleotide or deoxyribonucleotide monomers linked by phosphodiesterbonds, or by analogues linked by methyl phosphonare, phosphorothionateor other bonds. They may also comprise monomer moieties which havealtered base structures or other modifications, but which still retainthe ability to bind to naturally occurring transcript RNA structures.Such polynucleotides may be prepared by methods well-known in the art,for example, by using commercially available machines and reagents suchas those available from Perkin-Elmer/Applied Biosystems (Foster City,Calif.). For example, polynucleotides specific to a targeted transcriptare synthesized according to standard methodology. Phosphorothionatemodified DNA polynucleotides typically are synthesized on automated DNAsynthesized on automated DNA synthesizers available from a variety ofmanufacturers. These instruments are capable of synthesizing nanomoleamounts of polynucleotides as long as 100 nucleotides. Shorterpolynucleotides synthesized by modern instruments are often suitable foruse without further purification. If necessary, polynucleotides may bepurified by polyacrylamide gel electrophoresis or reverse phasechromatography. See, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Vol. 2, Chapter 11, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Phosphodiester-linked polynucleotides are particularly susceptible tothe action of nucleases in serum or inside cells, and therefore, in apreferred embodiment, the polynucleotides of the present invention arephosphothionate or methyl phosphonate-linked analogues, which have beenshown to be nuclease resistant. Persons having ordinary skill in the artcan easily select other linkages for use in this invention. Thesemodifications also may be designed to improve cellular uptake andstability of the polynucleotides.

An appropriate carrier for administration of a polynucleotide caninclude, for example, vectors, antibodies, pharmacologic compositions,binding or homing proteins, or viral delivery systems to enrich for thesequence into the target cell or tissue. A polynucleotide of the presentinvention can be coupled to, for example, a binding protein whichrecognizes endothelial cells or tumor cells. Following administration, apolynucleotide of the present invention can be targeted to a recipientcell or tissue such that enhanced expression of, for example, cytokines,transcription factors, G-protein coupled receptors, tumor suppressorproteins, and apoptosis initiation proteins can occur.

Delivery of antisense molecules and the like can be achieved using arecombinant expression vector such as a chimeric virus or a colloidaldispersion system. Various viral vectors which can be utilized for genetherapy as taught herein include adenovirus, herpes virus, vaccinia orpreferably an RNA virus such as a retrovirus. A number of the knownretroviruses can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and generated. By inserting apolynucleotide sequence of interest into the viral vector, along withanother gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is target specific. Retroviralvectors can be made target specific by inserting, for example, apolynucleotide encoding a sugar, a glycolipid or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome to allow target specificdelivery of the retroviral vector containing the antisensepolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. These assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enable the packaging mechanism to recognize anRNA transcript for encapidation. Helper cell lines which have deletionsof the packaging signal include but are not limited to ψ2, PA317 andPA12. These cell lines produce empty virions, since no genome ispackaged. If a retroviral vector is introduced into such cells in whichthe packaging signal is intact, but the structural genes are replaced byother genes of interest, the vector can be packaged and vector virionproduced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,poi, and env, by conventional calcium phosphate transfection. Thesecells are then transfected with the vector plasmid containing the genesof interest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for antisense molecules is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrance vesicles which are usefulas delivery systems in vivo and in vitro. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form. In order for a liposome to be an effective gene transfervehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation.

Small Molecule Inhibitors. The present invention further provides amethod in which small molecules which inhibit the activity of the CTGFfragment of the present invention are identified and utilized.

Identifying small molecules that inhibit the CTGF fragment activity canbe conducted by various screening techniques. For screening thecompounds, the assay will provide for a detectable signal associatedwith the binding of the compound to a protein or cellular target.Depending on the nature of the assay, the detectable signal may be lightabsorbance or emission, plaque formation, or other convenient signal.The result may be qualitative or quantitative.

For screening the compounds for specific binding, various immunoassaysmay be employed for detecting human (or primate) antibodies bound to thecells. Thus, one may use labeled anti-hlg, e.g., anti-hlgM, hlgG orcombinations thereof to detect specifically bound human antibody of thegalactosyl epitope. Various labels can be used such as radioisotopes,enzymes, fluorescers, chemiluminescers, particles, etc. There arenumerous commercially available kits providing labeled anti-hlg, whichmay be employed in accordance with the manufacturer's protocol.

Various protocols may be employed for screening a library of chemicalcompounds. To some degree, the selection of the appropriate protocolwill depend upon the nature of the preparation of the compounds. Forexample, the compounds may be bound to individual particles, pins,membranes, or the like, where each of the compounds is segregatable. Inaddition, the amount of compound available will vary, depending upon themethod employed for creating the library. Furthermore, depending uponthe nature of the attachment of the compound to the support, one may beable to release aliquots of a compound, so as to carry out a series ofassays. In addition, the manner in which the compounds are assayed willbe affected by the ability to identify the compound which is shown tohave activity.

Where the compounds are individually on a surface in a grid, so that ateach site of the grid one knows what the composition is, one can providea cellular lawn which is similarly organized as a grid and may be placedin registry with the compounds bound to the solid surface. Once the lawnand solid substrate are in registry, one may release the compounds fromthe surface in accordance with the manner in which the compounds areattached. After sufficient time for the compounds to bind to theproteins on the cellular surface, one may wash the cellular lawn toremove non-specifically bound compounds. One or more washings may beinvolved, where the washings may provide for varying degrees ofstringency, depending upon the desired degree of affinity. After thewashings have been completed, mammalian blood or plasma may then beadded and incubated for sufficient time for cytotoxicity. The plasma orblood may then be removed and plaques observed, where the nature of thecompound may be determined by virtue of the position in the grid. Ofcourse, the plasma or blood should be free of any components which wouldnaturally kill the cells of the lawn.

Since the preparative process may be repeated, one could prepare aplurality of solid substrates, where the same compounds are prepared atthe comparable sites, so that the screening could be repeated with thesame or different cells to determine the activity of the individualcompounds.

In some instances, the identity of the compound can be determined by anucleic acid tag, using the polymerase chain reaction for amplificationof the tag. See, for example, WO93/20242. In this instance, thecompounds which are active may be determined by taking the lysate andintroducing the lysate into a polymerase chain reaction mediumcomprising primers specific for the nucleic acid tag. Upon expansion,one can sequence the nucleic acid tag or determine its sequence by othermeans, which will indicate the synthetic procedure used to prepare thecompound.

Alternatively, one may have tagged particles where the tags arereleasable from the particle and provide a binary code which describesthe synthetic procedure for the compounds bound to the particle. See,for example, Ohlmeyer, et al., PNAS USA (1993) 90:10922. These tags canconveniently be a homologous series of alkylene compounds, which can bedetected by gas chromatography-electron capture. Depending upon thenature of the linking group, one may provide for partial release fromthe particles, so that the particles may be used 2 or 3 times beforeidentifying the particular compound.

While for the most part libraries have been discussed, any large groupof compounds can be screened analogously, so long as the CTGF epitopecan be joined to each of the compounds. Thus, compounds from differentsources, both natural and synthetic, including macrolides,oligopeptides, ribonucleic acids, dendrimers, etc., may also be screenedin an analogous manner.

Formation of a plaque in the assay demonstrates that binding of themember of the library to the cell, usually a surface protein, does notinterfere with the CTGF epitope binding to an antibody, that the immunecomplex is sufficiently stable to initiate the complement cascade, andthat the member has a high affinity for the target

Other screening methods for obtaining small molecules that modulate theactivity of CTGF fragments of the present invention are disclosed in PCTPublication WO 9813353.

Pharmaceutical Formulations and Routes of Administration

In order to identify small molecules and other agents useful in thepresent methods for treating or preventing a renal disorder bymodulating the activity and expression of CTGF, CTGF and biologicallyactive fragments thereof can be used for screening therapeutic compoundsin any of a variety of screening techniques. Fragments employed in suchscreening tests may be free in solution, affixed to a solid support,borne on a cell surface, or located intracellularly. The blocking orreduction of biological activity or the formation of binding complexesbetween CTGF and the agent being tested can be measured by methodsavailable in the art.

Other techniques for drug screening which provide for a high throughputscreening of compounds having suitable binding affinity to CTGF, or toanother target polypeptide useful in modulating, regulating, orinhibiting the expression and/or activity of CTGF, are known in the art.For example, microarrays carrying test compounds can be prepared, used,and analyzed using methods available in the art. See, e.g., Shalon, D.et al., 1995, PCT Application No. WO95/35505, Baldeschweiler et al.,1995, PCT Application No. WO95/251116; Brennan, T. M. et al., 1995, U.S.Pat. No. 5,474,796; Heller, M. J. et al., 1997, U.S. Pat. No. 5,605,662.

Identifying small molecules that modulate CTGF activity can also beconducted by various other screening techniques, which can also serve toidentity antibodies and other compounds that interact with CTGF and canbe used as drugs and therapeutics in the present methods. See, e.g.,Enna, S. J. et al., eds., 1998. Current Protocols in Pharmacology, JohnWiley and Sons. Assays will typically provide for detectable signalsassociated with the binding of the compound to a protein or cellulartarget. Binding can be detected by, for example, fluorophpres, enzymeconjugates, and other detectable labels well-known in the art. See,e.g., Enna et al. The results may be qualitative or quantitative.

For screening the compounds for specific binding, various immunoassaysmay be employed for detecting, for example, human or primate antibodiesbound to the cells. Thus, one may use labeled anti-Mg, e.g., anti-hlgM,hlgG or combinations thereof to detect specifically bound human antibodyof the galactosyl epitope. Various labels can be used such asradioisotopes, enzymes, fluorescers, chemiluminescers, particles, etc.There are numerous commercially available kits providing labeledanti-Mg, which may be employed in accordance with the manufacturer'sprotocol.

For screening the compounds for cytotoxic effects, a wide variety ofprotocols may be employed to ensure that one has the desired activity.One will normally use cells, which may be naturally occurring ormodified, cell lines, or the like. The cells may be prokaryotic oreukaryotic. For example, if one is interested in a pathogen, where itdoes not matter to which epitope the compound conjugate binds, one cancombine the pathogenic cells with each of the compounds in the presenceof an antibody dependent cytotoxic system to determine the cytotoxiceffect. One may perform this assay either prior to or subsequent todetermining the effect of the various candidate compounds on cells ofthe host to whom the compound would be administered. In this way, onewould obtain a differential analysis between the affinity for thepathogenic target and the affinity for host cells which might beencountered, based on the mode of administration.

In some situations, one would be interested in a particular cellularstatus, such as an activated state, as may be present with T cells inautoimmune diseases, transplantation, and the like. In this situationone would first screen the compounds to determine those which bind tothe quiescent cell, and as to those compounds which are not binding tothe quiescent cells, and screen the remaining candidate compounds forcytotoxicity to the activated cells. One may then screen for other cellspresent in the host which might be encountered by the compounds todetermine their cytotoxic effect. Alternatively, one might employ cancercells and normal cells to determine whether any of the compounds havehigher affinity for the cancer cells, as compared to the normal cells.Again, one could screen the library of compounds for binding to normalcells and determine the effect. Those compounds which are not cytotoxicto normal cells could then be screened for their cytotoxic effect tocancer cells. Even where some cytotoxicity exists for normal cells, inthe case of cancer cells, where there is a sufficient differentiation incytotoxic activity, one might be willing to tolerate the lowercytotoxicity for normal cells, where the compound is otherwise shown tobe effective with cancer cells.

Instead of using cells which are obtained naturally, one may use cellswhich have been modified by recombinant techniques. Thus, one may employcells which can be grown in culture, which can be modified byupregulating or downregulating a particular gene. In this way, one wouldhave cells that differ as to a single protein on the surface. One couldthen differentially assay the library as to the effect of members of thelibrary on cells for which the particular protein is present or absent.In this way, one could determine whether the compound has specificaffinity for a particular surface membrane protein as distinct from anyof the proteins present on the surface membrane.

One may differentiate between cells by using antibodies binding to aparticular surface membrane protein, where the antibodies do notinitiate the complement dependent cytotoxic effect, for example, usingdifferent species, isotypes, or combinations thereof. By adding theantibodies, blocking antisera or monoclonal antibodies, to one portionof the cells, those cells will not have the target protein available forbinding to the library member. In this way one creates comparative cellswhich differ in their response based on the unavailability in one groupof a single protein. While antibodies will usually be the mostconvenient reagent to use, other specific binding entities may beemployed which provide the same function.

For use in the assay to determine binding, one may use anantibody-dependent cytotoxic system. One could use synthetic mixtures ofthe ingredients, where only those components necessary for the cytotoxiceffect are present. This may be desirable where components of blood orplasma may adversely affect the results of the assay.

Also, while a cellular lawn is an extremely convenient way to screenlarge numbers of candidates, other techniques may also find use. Thesetechniques include the use of multiwell plates, and the various devicesused for the preparation of the combinatorial library, such as pins, teabags, etc. One may grow the cells separately in relation to the natureof the various devices, where the device may then be contacted with thecells or have the cells grown on the device. The device may be immersedin an appropriate culture, seeded with the cells, or otherwise providedfor contact between the cells and the candidate compound. After addingthe cytotoxic agent, one may then analyze for lysis in a variety ofways. For example, FACS may be used for distinguishing between live anddead cells, sup 51 Cr release may be employed, or detection of anintracellular compound in the supernatant, may serve to detect activecompounds.

In addition, one may wish to know whether the compound has agonist orantagonist activity. The subject assay techniques provide for a rapidway for determining those compounds present in the library which bind tothe target protein. Once, one has substantially narrowed the number ofcandidate compounds, one can use more sophisticated assays for detectingthe activity of the compound itself. In this way, one can perform arapid screen to determine binding affinity and specificity, followed bya more intensive screen to determine activity. Various techniques existfor determining activity, where the cells may be modified, so that amarker gene will be activated which will provide for a detectablesignal. Conveniently, the signal may be associated with production of adye, the production of a surface membrane protein which can be detectedwith labeled antibodies, or the secretion of a protein which can bedetected in the supernatant by any of a variety of techniques. Forexample, the gene that is expressed may be luciferase modified to have aleader sequence so as to be secreted, whereby the supernatant can thenbe screened for light generation formation by using an appropriatesubstrate.

Various protocols may be employed for screening the library. To somedegree, this will depend upon the nature of the preparation of thecompounds. For example, the compounds may be bound to individualparticles, pins, membranes, or the like, where each of the compounds issegregatable. In addition, the amount of compound available will vary,depending upon the method employed for creating the library.Furthermore, depending upon the nature of the attachment of the compoundto the support, one may be able to release aliquots of a compound, so asto carry out a series of assays. In addition, the manner in which thecompounds are assayed will be affected by the ability to identify thecompound which is shown to have activity.

Where the compounds are individually on a surface in a grid, so that ateach site of the grid one knows what the composition is, one can providea cellular lawn which is similarly organized as a grid and may be placedin registry with the compounds bound to the solid surface. Once the lawnand solid substrate are in registry, one may release the compounds fromthe surface in accordance with the manner in which the compounds areattached. After sufficient time for the compounds to bind to theproteins on the cellular surface, one may wash the cellular lawn toremove non-specifically bound compounds. One or more washings may beinvolved, where the washings may provide for varying degrees ofstringency, depending upon the desired degree of affinity. After thewashings have been completed, mammalian blood or plasma may then beadded and incubated for sufficient time for cytotoxicity. The plasma orblood may then be removed and plaques observed, where the nature of thecompound can be determined by virtue of the position in the grid. Theplasma or blood can be free of any components that would naturally killthe cells of the lawn.

Since the preparative process may be repeated, one could prepare aplurality of solid substrates, where the same compounds are prepared atthe comparable sites, so that the screening could be repeated with thesame or different cells to determine the activity of the individualcompounds. In some instances, the identity of the compound can bedetermined by a nucleic acid tag, using the polymerase chain reactionfor amplification of the tag. See, e.g., PCT Application No. WO93/20242.In this instance, the compounds that are active may be determined bytaking the lysate and introducing the lysate into a polymerase chainreaction medium comprising primers specific for the nucleic acid tag.Upon expansion, one can sequence the nucleic acid tag or determine itssequence by other means, which will direct the selection of theprocedure is used to prepare the compound.

Alternatively, one may have tagged particles where the tags arereleasable from the particle and provide a binary code that describesthe synthetic procedure for the compounds bound to the particle. See,e.g., Ohlmeyer, et al., 1993, PNAS 90:10922. These tags can convenientlybe a homologous series of alkylene compounds, which can be detected bygas chromatography-electron capture. Depending upon the nature of thelinking group, one may provide for partial release from the particles,so that the particles may be used 2 or 3 times before identifying theparticular compound.

While for the most part libraries have been discussed, any large groupof compounds can be screened analogously, so long as the CTGF epitopecan be joined to each of the compounds. Thus, compounds from differentsources, both natural and synthetic, including macrolides,oligopeptides, ribonucleic acids, dendrimers, etc., may also be screenedin an analogous manner.

Formation of a plaque in the assay demonstrates that binding of themember of the library to the cell, usually a surface protein, does notinterfere with the CTGF epitope binding to an antibody, that the immunecomplex is sufficiently stable to initiate the complement cascade, andthat the member has a high affinity for the target.

The subject methodology can be used in any situation where one has acellular target to be killed, particularly those cellular targets havinglow or no CTGF epitope. Thus, the cellular target may be a prokaryote,which is pathogenic. Various organisms include, for example,microbacterium, Yersinia, Pseudomonas, Bordetella pertussis, Treponemapallidum, Neisseria gonorrhoea, Streptococcus, Hemophilus influenza,etc. Other pathogens include eukaryotes, particularly fungi, such asCandida, Histoplasma, etc., and protozoa, e.g., Giardia. In addition,viruses which provide for surface membrane proteins in infected cells,can also be the target of the subject compounds, where the cells thatare screened have been vitally infected.

Host cells may also serve as targets, where the cells are eitherabnormal or act in an adverse way to the host or treatments of the host.For example, cancerous tissues which can be distinguished from normaltissue can serve as a target for the subject compounds. T or B cellsassociated with autoimmune diseases or associated with GVHD ortransplant rejection may also serve as targets. Aberrant cells,regardless of their nature, so long as they can be distinguished fromnormal cells, may also serve as targets. Thus, psoriatic lesions,lymphoma cells, bacterial, fungal, parasitic, virus infected cells, maybe targets of the subject products. Also, where one wishes to ablate aportion of cells, without removal of all of the cells, such as cellsexpressing a differentiation marker such as T cell subsets, activatedplatelets, endothelial cells, hormone or cytokine receptor expressingcells, the subject compounds may find application.

Other screening methods for obtaining small molecules that modulate theactivity of CTGF can be found, for example, PCT Application No. WO9813353.

Compounds/Molecules. The present invention provides methods for treatingand preventing disorders associated with kidney fibrosis by modulating,regulating, or inhibiting the activity of CTGF. These methods cancomprise the administration of a therapeutically effective amount of acompound that blocks the binding interactions or blocks enzymes involvedin the signal transduction pathway of CTGF. More specifically, thepresent invention provides a method for inhibiting the activity of CTGFby administering compounds that block the induction of CTGF.

Compounds that modulate CTGF gene expression and/or CTGF activity in themethod of the invention include agents which cause an elevation incyclic nucleotides in the cell. Other compounds that may block theinduction of CTGF according to the methods of the present invention, maybe identified using the screening methods described above.

In one embodiment, the invention provides a method of identifying acompound or an agent which modulates the activity, e.g., production ofextracellular matrix proteins, induction of myofibroblastdifferentiation, induction of collagen synthesis, of a CTGF fragment,e.g., a fragment encoded by exons 2 and 3 as set forth in FIG. 3. Themethod includes contacting an agent of interest, such as a peptide,small molecule, polypeptide, peptidomimetic, with the cells (e.g.,fibroblasts) and a CTGF fragment known to have the desired activity,e.g., production of collagen, and measuring the ability of the cells toproduce collagen by any means known to one of skill in the art (seeEXAMPLES). The ability of the cells to produce collagen is then comparedto the ability of a suitable control population of cells to producecollagen in the absence of the agent or compound.

The term “agent” or “compound” as used herein describes any molecule,e.g., a protein, polypeptide, or pharmaceutical, with the capability ofaffecting an activity of a CTGF fragment as described herein. The agentcan be anything known or suspected of being capable of affecting thecells. The agent includes peptide fragments of CTGF polypeptide. Theagents include synthetic chemical agents, biochemical agents, cells,extracts, homogenates and conditioned medium. The test agent may also bea combinatorial library for screening a plurality of compounds.Compounds identified in the method of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence, such as PCR, oligomerrestriction (Saiki et al., Bio/Technology 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,Proc. Natl. Acad Sci. USA 80:278, 1983), oligonucleotide ligation assays(OLAs) (Landegren et al., Science 241:1077, 1988), and the like.Molecular techniques for DNA analysis have been reviewed (Landegren etal., Science 242:229-237, 1988).

Candidate agents encompass numerous chemical classes. They can beorganic molecules, preferably small organic compounds having a molecularweight of more than 50 and less than about 2,500 Daltons. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amino, carbonyl, hydroxyl or carboxyl group, preferably atleast two functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding, but not limited to: peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Candidate agents can be polypeptides, orpolypeptides produced by site-directed or random mutagenesis of asynthetic or naturally occurring nucleic acid sequence.

In yet a further embodiment of the present invention, the methodprovides for the administration of molecules that interrupt thepost-translational modification of full length CTGF or block theactivation of an inactive precursor of CTGF. As discussed herein,exposure of mesangial cells to TGF-β resulted in the marked appearanceof additional bands at 28-30 kDa which correspond in size to thecarboxy- and amino-terminal halves of the full length CTGF molecule. Asdisclosed above, TGF-β treatment may result in the production ofproteases or other factors capable of cleaving the full-length molecule.Molecules that inhibit CTGF activity may be identified using thescreening methods provided herein.

Pharmaceutical Formulations and Routes of Administration

Routes of Administration. The compositions comprising CTGF modulators,i.e., the antibodies, antisense oligonucleotides, small molecules andother compounds as described herein can be administered to a humanpatient per se, or in pharmaceutical compositions comprising, whereappropriate, suitable carriers or excipients. The present inventioncontemplates methods of treatment in which agents that modulate orregulate the expression or activity of CTGF or fragments thereof areadministered to a patient in need, in amounts suitable to treat orprevent the activity or expression of the CTGF fragment The presentmethods of treatment and prevention can comprise administration of aneffective amount of the agent to a subject which is preferably amammalian subject, and most preferably a human subject. In a preferredembodiment, the subject mammal and the agent administered are ofhomologous origin. Most preferably, the subject and the agentadministered are human in origin.

An effective amount can readily be determined by routine experiment, ascan the most effective and convenient route of administration and themost appropriate formulation. Various formulations and drug deliverysystems are available in the art. See, e.g., Gennaro, A. R., ed., 1990,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton Pa. Suitable routes of administration may, for example, includeoral, rectal, transmucosal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Thecomposition may be administered in a local rather than a systemicmanner.

The pharmaceutical compositions of the present invention may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes. Asnoted above, the compositions of the present invention can include oneor more physiologically acceptable carriers such as excipients andauxiliaries which facilitate processing of active molecules intopreparations for pharmaceutical use. Proper formulation is dependentupon the route of administration chosen.

For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject. The compoundsmay also be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, for example,gelatin, for use in an inhaler or insufflator may be formulated. Thesetypically contain a powder mix of the compound and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions of agents that effect the activity of CTGF or fragmentsthereof, in water-soluble form.

Suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions of the present invention may also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical carriers for the hydrophobic molecules of the inventioncould include co-solvent systems comprising, for example, benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer, and anaqueous phase. The co-solvent system may be the VPD co-solvent system.VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolarsurfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made upto volume in absolute ethanol. The VPD co-solvent system (VPD:5W)consists of VPD diluted 1:1 with a 5% dextrose in water solution. Thisco-solvent system is effective in dissolving hydrophobic compounds andproduces low toxicity upon systemic administration. Naturally, theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics. Furthermore, theidentity of the co-solvent components may be varied. For example, otherlow-toxicity nonpolar surfactants may be used instead of polysorbate 80,the fraction size of polyethylene glycol may be varied, otherbiocompatible polymers may replace polyethylene glycol, e.g. polyvinylpyrrolidone, and other sugars or polysaccharides may substitute fordextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing sustained-release systems, such as semi-permeable matrices ofsolid hydrophobic polymers containing the effective amount of thecomposition to be administered. Various sustained-release materials areestablished and available to those of skill in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Effective Dosage. For any composition used in the present methods oftreatment, a therapeutically effective dose can be estimated initiallyusing a variety of techniques well-known in the art. For example, in acell culture assay, a dose can be formulated in animal models to achievea circulating concentration range that includes the IC₅₀ as determinedin cell culture. Where inhibition of CTGF activity is desired, forexample, the concentration of the test compound which achieves ahalf-maximal inhibition of CTGF activity can be determined. Dosageranges appropriate for human subjects can be determined using dataobtained from cell culture assays and other animal studies.

A therapeutically effective dose refers to that amount of the moleculethat results in amelioration of symptoms or a prolongation of survivalin a subject. Toxicity and therapeutic efficacy of such molecules can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio of toxic to therapeuticeffects is the therapeutic index, which can be expressed as the ratioLD₅₀/ED₅₀. Molecules which exhibit high therapeutic indices arepreferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage will be chosen in view of the specifics of asubject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to modulate orregulate CTGF activity as desired, i.e. minimal effective concentration(MEC). The MEC will vary for each compound but can be estimated from,for example, in vitro data, such as the concentration necessary toachieve 50-90% activity of CTGF to induce bone growth using the assaysdescribed herein.

Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Compositions should beadministered using a regimen which maintains plasma levels above the MECfor about 10-90% of the duration of treatment, preferably about 30-90%of the duration of treatment, and most preferably between 50-90%. Incases of local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent ona number of factors, including, but not limited to, the particularsubject's weight, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

Packaging. The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of disorders or diseases in which cartilage or bone induction,wound healing, neuroprotection, kidney fibrosis, diabetes, or the likeis desired.

EXAMPLES

The following examples are provided solely to illustrate the claimedinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

Example 1 CTGF Fragments Stimulate Extracellular Matrix Synthesis

To prepare CTGF fragments, human recombinant CTGF (full length) wasdigested by chymotrypsin to render one CTGF fragment. Recombinant CTGFfragments were also produced by expressing either or both exon 2 andexon 3 of CTGF. A continuous line of cultured normal rat kidney (NRK)fibroblasts, designated as clone NRK-49F, were obtained from theAmerican Type Culture Collection (ATCC) to produce cell cultures. Humanforeskin fibroblasts were established from explant cultures. Cellcultures were maintained in Dulbecco's modified eagle media (DME)containing 2.5% fetal bovine serum and 2.5% Nu-Serum I (CollaborativeBiomedical Products, Bedford, Mass.) and passaged prior to confluence.

To examine fibroblast collagen synthesis, growth-arrested monolayers ofNRK and human foreskin fibroblasts were prepared by seeding 10,000cells/well in 48 well plates and allowing the cells to grow toconfluence in 5 to 7 days in DME and 2.5% fetal bovine serum/Nu-Serum.Fibroblast monolayers were then serum-starved in DME containing 25 mMHEPES and ITS premix (Collaborative Biomedical) for 1 to 8 days.Ascorbic acid (50 mg/ml) and biological agents (CTGF fragments) werethen added. Collagen synthesis was assessed by measuring ³H-prolineincorporation into pepsin resistant salt precipitatedextracellular/cell-surface associated collagen using a quantitativeassay for the terminal 24 hours of the 48 hour treatment. As set forthin FIG. 2, the CTGF fragments comprising exons 2 and 3 of CTGFstimulated collagen synthesis in NRK fibroblasts with 1 ng/ml of TGF-b.In contrast, the carboxyl terminal CTGF did not stimulate collagensynthesis.

Example 2 CTGF Fragments Induce Myofibroblast Differentiation

The CTGF fragments and cell cultures were prepared as described above.To examine induction of myofibroblasts by CTGF fragments,growth-arrested monolayers of NRK for foreskin fibroblasts were preparedand treated as described above. Following treatments, 48-well platemonolayers were washed twice with TBS and fixed in methanol at −20degrees C. for 10 minutes before processing for immunohistologicaldetection of alpha-smooth muscle. Immunohistological detection wasconducted. Following fixation, cell monolayers were washed twice withTBS and blocked for 30 minutes with 10% horse serum/2% milk in TBS. Thecells were then incubated for 1 hour with monoclonal mouse antialpha-smooth muscle actin IgG (Clone 1A4, Sigma Chemical) at a 1:200dilution in horse serum/milk/TBS. Following three washes with TBS, cellmonolayers were then incubated for 1 hour with biotinlyated horseanti-mouse IgG (Vector Labs, Burlingame, Calif.) at a 1:200 dilution inhorse serum/milk/TBS. The cell monolayers were then washed and incubatedfor 30 minutes with an alkaline phosphate conjugated streptvidin-biotincomplex (Dako, Glostrup, Denmark). Following washing, alkalinephosphatase was then visualized with a fast red substrate (Vector Red,Vector Labs) in the presence of 1 mM levamisole. Processed cellmonolayers were then examined under a microscope for red stainedalpha-smooth muscle actin positive myofibroblasts and the number ofmyofibroblasts per well were counted and selected microscopic fieldswere photographed.

As set forth in FIG. 1, the results from the assay for fibroblastcollagen synthesis are mirrored by the myoblast induction assay.Specifically, the CTGF fragments of the present invention were able toinduce myoblast differentiation, as compared to carboxyl terminal CTGFfragments, which were unable to induce such differentiation.

Example 3 Neutralizing Anti-CTGF Antibodies Block TGF-B Induced DNASynthesis, Collagen Synthesis, and Myofibroblast Induction

Specific anti-CTGF antibodies were raised against biologically activerecombinant human CTGF produced in a baculovirus expression system usingmethods known in the art. The antibodies were prepared in goats andtested for neutralization activity of CTGF directly or on TGF-inducedDNA or collagen synthesis in NRK fibroblasts. The goat antibodiesexhibited activity in the assays for neutralization of TGF-13 action. Inthese assays, the goat anti-CTGF antibodies were able to block DNAsynthesis. In addition, as demonstrated in FIG. 5, CTGF antibodies wereable to block collagen synthesis and myofibroblast formation induced byTGF-β. It was noted that the amount of antibody required to blockcollagen synthesis was significantly less than the amount needed toblock DNA synthesis. Both western Mot assay, and competition ELISAassays indicated that most of the antibodies in this preparation weredirected against the N-terminal domain of CTGF. This suggested to thatthe two domains might be responsible for stimulating differentbiological activities.

Example 4 Anti-CTGF Antibodies Specific for the N-Terminal Domain ofCTGF Selectively Block Collagen Synthesis

Domain specific anti-CTGF antibodies were prepared by affinitychromatography using purified N-terminal or C-terminal CTGF domains.These domains were prepared from intact CTGF by limited digestion withchymotrypsin. The domains were separated from each other by affinitychromatography on heparin sepharose. The N-terminal domain does not bindto heparin whereas the C-terminal domain of CTGF contains the heparinbinding activity and is retained on the heparin sepharose. These domainswere pure, having less than 0.1% contamination with intact CTGF based onwestern blot analysis. The individual domains were then coupled toAffigel 10 at a concentration of approximately 0.5 mg/ml of gel. Totalanti-CTGF IgG (goat) was then absorbed to the affinity resin, and thespecifically bound antibodies were eluted. These antibodies were thentested in western blots to determine the specificity of theirreactivity. IgG=s reactive with only the N-terminal domain or only theC-terminal domain of CTGF was isolated from the total pool usingtechniques known in the art. The antibodies were then tested inneutralization assays using CTGF. The results of these studies indicatedthat antibodies directed against the N-terminal domain of CTGFselectively inhibited collagen synthesis, but not DNA synthesis asdemonstrated in FIG. 6. In contrast, antibodies directed against the Cdomain of CTGF selectively inhibited DNA synthesis, but not collagensynthesis. The data indicated that different regions of the CTGFmolecule may be responsible for signaling different biologicalactivities. To confirm and extend these results, the biologicalactivities of the isolated domains with intact CTGF and with TGF-β, werecompared as set forth below.

Example 5 The CTGF N-Terminal Domain Stimulates Extracellular MatrixProduction

CTGF N-terminal and C-terminal domains were prepared using techniquesknown in the art. First, as described above, pure N-terminal andC-terminal domains were prepared by proteolytic digestion ofbiologically active intact CTGF using chymotrypsin, since it producedalmost exclusively intact N-terminal domains and C-terminal domains withno smaller fragments. A second method to generate pure C-terminal andN-terminal domains, entailed expressing only limited regions of the CTGFopen reading, which encoded only the C-terminal domain or only theN-terminal domain. This was accomplished by PCR amplification ofportions of the open reading frame, and introducing either a stop codonin the cysteine free region to produce only the N-terminal domain orcloning the portion of the open reading frame encoding only theC-terminal domain, beginning at the sequence AYRLED in the cysteine freeregion in to the baculovirus shuttle vector GP67. This produced achimeric protein containing a signal peptide from the GP67 virus genethat directed synthesis of the desired recombinant protein (or fragment)to the endoplasmic reticulum, thus ensuring secretion. Afterpurification, the isolated domains generated by the various methods werecompared in a bioassay with NRK fibroblasts. The results of thesestudies confirmed the previous observations with the domain specificanti-CTGF antibodies. The N-terminal domains produced by eitherproteolytic digestion of intact CTGF or by direct recombinant expressionwere fully active as inducers of collagen synthesis and myofibroblastinduction as indicated in FIG. 7 and FIG. 8. Conversely, the C-terminaldomains produced by either method were also fully active in the DNAsynthesis assay. The data demonstrated that the individual domains ofCTGF retained full biological activity, and can act independently ofeach other to stimulate specific biological effects on target cells. Atoptimal concentrations, the individual domains induced a biologicalresponse comparable to intact CTGF or TGF-13. This strongly indicatedthat mitogenic (DNA synthesis) and matrigenic (extracellular matrixsynthesis, such as collagen synthesis) activities of TGF-β are mediatedvia CTGF and its respective domains.

Other growth factors may be used with the CTGF fragments of the presentinvention to increase the inductive activity of CTGF on extracellularmatrix production. For example, the peptide growth factor, IGF-2, wasevaluated for its effect on collagen and myofibroblast phenotypeinductive activity of CTGF. Concentrations of 2 ng/ml or higher of IGF-2in the presence of CTGF resulted in a large increase in collagensynthesis and myofibroblast phenotype as shown in FIG. 9.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims. All references cited herein are incorporated by reference hereinin their entirety.

1. An antisense molecule that binds to a nucleic acid sequence encodinga fragment of CTGF polypeptide, wherein the fragment of CTGF has theability to induce extracellular matrix and/or collagen synthesis.
 2. Theantisense molecule of claim 1, wherein the fragment of CTGF comprises anamino acid sequence selected from the group consisting of: (a) residue24 through 95 of SEQ ID NO:4; (b) residue 96 through 180 of SEQ ID NO:4;and (c) residue 24 through 180 of SEQ ID NO:4.
 3. A method for treatinga CTGF-associated disease or disorder comprising administering to asubject in need the antisense molecule of claim
 1. 4. The method ofclaim 3, wherein the disease or disorder is a fibroproliferativedisease/disorder.
 5. The method of claim 3, wherein the disease ordisorder is selected from the group consisting of kidney fibrosis,scleroderma, pulmonary fibrosis, liver fibrosis, arthritis, hypertropicscarring, atherosclerosis, diabetic nephropathy and retinopathy,hypertension, kidney disorders, angiogenesis-related disorders, skinfibrotic disorders, and cardiovascular disorders.